Diagnostic blood test for sarcoidosis

ABSTRACT

Sarcoidosis is a multisystem disease characterized by granulomatous inflammation in affected organs. We have developed a blood test using mycobacterial catalase-peroxidase that has a high positive predictive value for confirming a diagnosis of sarcoidosis.

CROSS-REFERENCE TO PRIOR FILED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/924,410 filed Jan. 7, 2014, which is incorporated herein in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos. P50HL107185 and R01 HL083870 awarded by the National Heart Lung and BloodInstitute (NHLBI). The government has certain rights in the invention.

TECHNICAL FIELD

This present disclosure generally relates to methods and reagents fordetecting Sarcoidosis.

BACKGROUND OF THE INVENTION

Sarcoidosis is a multisystem disease characterized by granulomatousinflammation in affected organs. There are no useful biomarkers toconfirm a diagnosis of sarcoidosis. A consensus among the medicalcommunity is that there is no blood test with sufficient specificity andsensitivity to be useful as a diagnostic test. Confirmation of adiagnosis of sarcoidosis in most cases requires a biopsy with itsattendant risks and costs.

Using a proteomic approach, mKatG has been identified as a tissueantigen and target of the immune response in sarcoidosis (J. Exp. Med.(2005) 201:755-67; U.S. Pat. Appl. Pub. No. US 2009/0175798). Animmunoassay was used to identify T cell responses to mKatG and thisallowed the detection of a secreted cytokine, interferon-gamma (INFγ),in response to mKatG. However, this immunoassay, using INFγ-ELISPOT,lacked the ability to distinguish between individuals with sarcoidosisand individuals with tuberculosis (TB) infection from Mycobacteriumtuberculosis with or without a positive purified protein derivative(PPD) skin test (also called a tuberculin skin test) or individualspreviously vaccinated with BCG (Bacillus Calmette-Guérin), derived froman attenuated strain of Mycobacterium bovis. Both of those conditionsgave positive reactions to the INFγ-ELISPOT assay (T cell responses tomKatG in 50% of sarcoidosis patients and 50-60% BCG+ or PPD+ subjects).(J. Immunol. (2008) 181:8784-96). In addition, this assay could notdistinguish sarcoidosis from individuals with non-tuberculousmycobacterial infection. All of these ailments have diseasemanifestations that can mimic or overlap with manifestations ofsarcoidosis, and thus, these ailments must be excluded before adiagnosis of sarcoidosis can be confirmed.

What is needed is a safer protocol with adequate specificity andsensitivity to assist clinicians in confirming a diagnosis ofsarcoidosis.

SUMMARY OF THE INVENTION

Specific microbial proteins, including mycobacterial catalase-peroxidaseprotein, are found in sarcoidosis tissues and are a target of the immunesystem of patients with sarcoidosis. Accordingly, diagnostic andprognostic methods are provided, comprising the use of mycobacterialcatalase peroxidase protein or derivatives or variants thereof. Theprotein may be synthesized by recombinant or chemical methods.

The methods may be incorporated into any test format or device suitablefor the practice of the methods. Also provided are kits, reagents, etc.for the practice of the methods.

Described herein is a blood test that has a high positive predictivevalue for confirming a diagnosis of sarcoidosis. The blood test uses, ina first embodiment, Mycobacterium tuberculosis catalase-peroxidase(mKatG), and a commercially available mixture of mycobacterial proteinscalled purified protein derivative (PPD) to stimulate whole blood cellsto release an inflammatory cytokine called interferon gamma (INFγ). TheINFγ levels from each stimulatory or control condition are measured andthe values are applied to an algorithm, which provides data that havebeen shown to have a high positive predictive value for sarcoidosis. Thealgorithm is used to predict sarcoidosis, as distinguished from latentor active tuberculosis infection with or without a positive PPD skintest, individuals with a previous vaccination with BacillusCalmette-Guérin (BCG), individuals with non-tuberculous mycobacterialinfection, or individuals with diseases other than sarcoidosis.

The invention is a blood test that can be used to assist in thediagnosis of sarcoidosis. This blood test requires the followingspecifications in order to operate as a diagnostic test for sarcoidosis:reagents mKatG and PPD purified to certain specifications and used in aspecific dose range, the details of which are set forth herein; reagentsmKatG, PPD, and a background (no stimulation) used in separateconditions; the use of endotoxin neutralizing agents in the mKatG andPPD conditions; the use of an assay to accurately measure levels of IFNγin plasma; the use of a defined algorithm that compares the results ofINFγ released in the background, mKatG and PPD conditions. The use of aT cell stimulation reagent as a positive control in a separate conditionto serve as a quality control measure and assist in the interpretationof whether an individual is capable of responding to the other testconditions (mKatG, PPD) but does not factor into the diagnosticalgorithm.

In this embodiment of the invention, the process is a method for aidingin the prediction of whether an individual has sarcoidosis, the methodcomprising:

-   (a) treating a first aliquot of blood from the individual as a    control having no added INFγ-releasing reagent;-   (b) contacting a second aliquot of blood from the individual with    fluid containing mKatG in an amount that is ≧0.1 mcg/ml;-   (c) contacting a third aliquot of blood from the individual with    fluid containing PPD in an amount that is ≧0.1 mcg/ml;-   (d) detecting the amount of INFγ in the aliquots;-   (e) calculating adjusted amounts of INFγ as amounts of INFγ in the    second and third aliquots minus the amount of INFγ in the first    aliquot; and-   (f) associating a prediction of sarcoidosis with the determination    that there is (1) an adjusted amount of INFγ for the second aliquot    of greater than 100 pg/ml as well as that there is (2) an adjusted    amount of INFγ for the second aliquot that is greater than the    adjusted amount of INFγ for the third aliquot.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise above, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Where a term isprovided in the singular, the inventor also contemplates the plural ofthat term. The nomenclature used herein and the procedures describedbelow are those well known and commonly employed in the art.

The singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

The terms “comprise” and “comprising” is used in the inclusive, opensense, meaning that additional elements may be included.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof, amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing. The names of the naturalamino acids are abbreviated herein in accordance with therecommendations of IUPAC-IUB.

The term “antigenic fragment” refers to a polypeptide fragment or regionof a polypeptide that is able to elicit an immune response. An “immuneresponse” refers to the reaction of a subject to the presence of anantigen, which may include at least one of the following: makingantibodies, developing immunity, developing hypersensitivity to theantigen, and developing tolerance.

The term “condition” when used with reference to the assay method refersto a sample measurement obtained under particular experimentalconditions that differ from the experimental conditions of anothersample. Thus when aliquots of patient's blood are exposed to differentreagents and then measured for interferon gamma, each of these differentmeasurements obtained as a result of exposure to different reagents orto a control are referred to as a condition; e.g. the mKatG condition,the PPD condition, the background condition.

“Derivative” refers to the chemical modification of a polypeptidesequence, or a polynucleotide sequence. Chemical modifications of apolynucleotide sequence may include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A derivative polynucleotideencodes a polypeptide which retains at least one biological orimmunological function of the natural molecule. A derivative polypeptideis one modified by glycosylation, pegylation, or any similar processthat retains at least one biological or immunological function of thepolypeptide from which it was derived.

“EU” refers to endotoxin units. Because endotoxin molecular weight mayvary a great deal (10,000 to 1,000,000 Daltons), endotoxin is measuredin Endotoxin Units (EU). One EU equals approximately 0.1 to 0.2nanograms of E. Coli lipopolysaccharide. One assay for measurement ofendotoxin is the Limulus amebocyte lysate (LAL) assay. Currently thereare at least four forms of the LAL assay, each with differentsensitivities. The LAL gel clot assay can detect down to 0.03 EU/mLwhile the LAL kinetic turbidimetric and chromogenic assays can detectdown to 0.005 EU/mL.

The term “microbial catalase or peroxidase protein” refers to anycatalase-peroxidase, catalase or peroxidase protein from a microbe, forexample, catalase-peroxidase, catalase or peroxidase proteins frommycobacterial species such as Mycobacterium tuberculosis andMycobacterium smegmatis, or other bacterial species such as Helicobacterpylori and Propionibacterium acnes.

The term “non-tuberculous mycobacteria” (NTM) refers to allmycobacterial species other than Mycobacterium tuberculosis (Mtb) andincludes many common mycobacteria that are closely related toMycobacterium tuberculosis.

The terms “polypeptide fragment” or “fragment,” when used in referenceto a particular polypeptide, refers to a polypeptide in which amino acidresidues are deleted as compared to the reference polypeptide itself,but where the remaining amino acid sequence is usually identical to thatof the reference polypeptide. Such deletions may occur at theamino-terminus or carboxy-terminus of the reference polypeptide, oralternatively both. Fragments typically are at least about 5, 6, 8 or 10amino acids long, at least about 14 amino acids long, at least about 20,30, 40 or 50 amino acids long, at least about 75 amino acids long, or atleast about 100, 150, 200, 300, 500 or more amino acids long. A fragmentcan retain one or more of the biological activities of the referencepolypeptide. In various embodiments, a fragment may comprise anenzymatic activity and/or an interaction site of the referencepolypeptide. In another embodiment, a fragment may have immunogenicproperties.

A “patient” or “subject” or “host” refers to either a human or non-humananimal.

The term “purified” refers to an object species that is the predominantspecies present (i.e., on a molar basis it is more abundant than anyother individual species in the composition). A “purified fraction” is acomposition wherein the object species comprises at least about 50percent (on a molar basis) of all species present. In making thedetermination of the purity of a species in solution or dispersion, thesolvent or matrix in which the species is dissolved or dispersed isusually not included in such determination; instead, only the species(including the one of interest) dissolved or dispersed are taken intoaccount. Generally, a purified composition will have one species thatcomprises more than about 80 percent of all species present in thecomposition, in other embodiments more than about 85%, 90%, 95%, 99% ormore of all species present. The object species may be purified toessential homogeneity (contaminant species cannot be detected in thecomposition by conventional detection methods) wherein the compositionconsists essentially of a single species. A skilled artisan may purify apolypeptide of the invention using standard techniques for proteinpurification in light of the teachings herein. Purity of a polypeptidemay be determined by a number of methods known to those of skill in theart, including for example, amino-terminal amino acid sequence analysis,gel electrophoresis and mass-spectrometry analysis.

The term “PPD” refers to a mixture of mycobacterial proteins known aspurified protein derivative. The term “ PPD+” refers to a positiveMantoux skin test for tuberculosis, which standardly consists of anintradermal injection of one tenth of a milliliter (mL) of PPDtuberculin.

“Recombinant protein”, “heterologous protein” and “exogenous protein”are used interchangeably to refer to a polypeptide which is produced byrecombinant DNA techniques, wherein generally, DNA encoding thepolypeptide is inserted into a suitable expression vector which is inturn used to transform a host cell to produce the heterologous protein.That is, the polypeptide is expressed from a heterologous nucleic acid.

“Vector” refers to a nucleic acid molecule capable of transportinganother nucleic acid to which it has been linked. One type of preferredvector is an episome, i.e., a nucleic acid capable of extra-chromosomalreplication. Preferred vectors are those capable of autonomousreplication and/or expression of nucleic acids to which they are linked.Vectors capable of directing the expression of genes to which they areoperatively linked are referred to herein as “expression vectors”. Ingeneral, expression vectors of utility in recombinant DNA techniques areoften in the form of “plasmids” which refer generally to circular doublestranded DNA loops, which, in their vector form are not bound to thechromosome. In the present specification, “plasmid” and “vector” areused interchangeably as the plasmid is a commonly used form of vector.However, as will be appreciated by those skilled in the art, theinvention is intended to include such other forms of expression vectorswhich serve equivalent functions and which become subsequently known inthe art.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention.

In describing alternative embodiments, the inclusion of variousembodiments is illustrative and is not intended to limit the inventionto those particular embodiments.

Diagnostic Blood Test

The diagnostic blood test for sarcoidosis uses new methodology whichimproves the diagnostic specificity for sarcoidosis. Prior art in thediagnostic field was unable to sufficiently distinguish between subjectswho had sarcoidosis and those subjects with active mycobacterial diseasefrom tuberculosis (TB) or non-tuberculous mycobacteria, or subjects whowere PPD (latent TB infection), or subjects who had been vaccinated forTB (BCG vaccination). The present inventive method distinguishessarcoidosis from these and other diseases that are not sarcoidosis.

The diagnostic blood test for sarcoidosis measures release of INFγ fromimmune cells in blood after contact of the blood cells with the purifiedreagents mKatG or PPD (PPD derived from M. tuberculosis) or aftercontact with various control conditions (e.g., no contacting reagent isadded or the contacting reagent is not expected to cause release ofINFγ). Measurement of INFγ release in response to these reagentsprovides a specific and sensitive assay. Purifying or neutralizingcontaminating endotoxins from the specific reagents mKatG or PPD reducesnon-specific responses of INFγ release. The diagnostic blood testseparately measures release of INFγ from immune cells in blood aftercontact of the blood cells with a T cell stimulating reagent (positivecontrol) to provide a quality control measure and an assessment of theoverall ability of the immune cells in the blood to respond to immunestimulating reagents.

The whole blood test aliquots are combined with test reagents or controlreagents and incubated to allow for measurable release of INFγ.Preferably, the incubation is about 12 hours, about 12-18 hrs, or about12-24 hours. Incubation periods longer than about 24 hours are feasiblebut not time efficient.

Any suitable method of measuring INFγ is envisioned. Suitability refersto an assay system that is accurate, sensitive, robust and reproducible.Sensitivity of about 4 pg/ml (or the equivalent in International Units(IU) established by using World Health Organization standards) would besuitable. The method should have the capability to recover and measureINFγ in complex fluids such as plasma and serum without interference byconfounding serum factors. Examples of measurement methods includeELISA, RIA and multiplex arrays.

The algorithm used in conjunction with the illustrative blood assaystates that sarcoidosis is indicated when two circumstances are met:First, the concentration of INFγ in mKatG-stimulated blood minus theconcentration of INFγ in blood without a stimulating reagent is greaterthan 100 pg/ml; second, the concentration of INFγ in mKatG-stimulatedblood is greater than the concentration of INFγ in PPD-stimulated blood.(Hereafter, for simplicity, the algorithm will use nomenclature denotingthe separate conditions such as mKatG or PPD to mean the concentrationof INFγ released in the respective condition measured in pg/ml. Thecondition of blood without a stimulating reagent will be denoted asbackground or bkd). Thus, sarcoidosis is indicated when: mKatG minusbkd>100 and mKatG>PPD.

The whole blood stimulation assay algorithm quite accurately predictspersons with sarcoidosis because in most cases the blood of thesepersons measures higher INFγ release for mKatG stimulation than PPDstimulation (mKatG>PPD) whereas persons who are PPD+, have had BCGvaccination or have active or latent mycobacterial (MTB ornon-tuberculous mycobacterial) disease almost always measure higher INFγfor PPD stimulation than for mKatG stimulation (PPD>mKatG). When testingthe blood of healthy subjects or those with disease other thansarcoidosis or mycobacterial disease, mKatG stimulation minus backgroundcondition (without stimulating reagent) is usually less than 100 pg/mlINFγ (mKatG minus bkd<100), but when mKatG minus background is higherthan 100 pg/ml, then PPD>mKatG.

In another embodiment, to adjust the algorithm based on differentlaboratory conditions, the algorithm can use diagnostic cut-off levels,thresholds, or variables that are determined by testing knownsarcoidosis and control subjects, such as shown in Table 1. Furthermore,the thresholds, diagnostic cut-off levels or variables for bothconditions of the algorithm can be determined by using standardstatistical tests, wherein the sensitivity and specificity of the assaycan be increased or decreased and the receiver operating characteristiccurves can be used to maximize the diagnostic power of the test indifferent populations. This is described in more detail below.

In one embodiment, control subjects that do not have sarcoidosis and donot have mycobacterial disease, such as those in the right columnslabeled 1-5, can be used to determine a threshold for the firstcondition of the algorithm. In this embodiment, the first condition ofthe algorithm is mKatG is greater than the threshold established bytesting such control subjects. To further illustrate the use of controlsubjects, under the laboratory conditions used to establish the data inTable 1, all healthy subjects tested had a mKatG normalized tobackground below the threshold of 100pg/ml. Thus, under these conditionsthe threshold was established at this concentration. Therefore, ifdifferent assay conditions were used with the same control subjects andthe same sensitivity and specificity was desired, a different thresholdcould be established.

The variable, Y, in the second condition of the algorithm can bedetermined by using known control samples, such as those in Table 1. Asfurther illustrated in Table 1, mycobacterial infected control subjects,represented by samples in the right columns labeled 9, 16, 21-28, 32-35,and 37, all have PPD greater than mKatG. Thus, in a preferredembodiment, a variable, Y, can be determined to increase or decrease thevalue of PPD based on the values detected under different laboratoryconditions, such as where different preparations of reagents are used.In another embodiment, Y adjusts the value to a number that is equal tomKatG minus PPD for such controls. Furthermore, non-sarcoidosis diseasecontrols, such as samples in the right columns labeled 1-8, or healthysubjects represented in Table 1 can be used to establish the variable,Y, for the second condition of the algorithm.

Both mKatG and PPD reagents tend to contain considerable endotoxin thatcause non-specific elevation in INFγ levels when stimulating wholeblood. Endotoxins are not protein antigens that induce adaptive B or Tcell immune responses through antigen-specific receptors. Rather, theystimulate the immune system through independent receptor systems foundon many types of cells

Whole blood stimulation by endotoxin leads to quite variable results inINFγ release between different individuals. Therefore, for the inventiveblood test, it is necessary that endotoxins are substantiallyneutralized as immune system stimulators or are substantially absentfrom PPD and mKatG preparations. This can be accomplished if the mKatGand PPD reagents are purified to lower levels of endotoxin by suitablemeans. As an example, Endotrap® columns accomplish this and have beenable to reduce endotoxin levels of PPD to less than 0.10 EU/μg protein.Other suitable means of purification for mKatG are detailed in U.S. Pat.Pub. No. US 2009/0175798.

Purification by any suitable means are envisioned such as byultrafiltration or various modes of chromatography (e.g., HPLC, reversephase HPLC or ion exchange).

Due to the charged nature of endotoxins, strong anion exchangechromatography is particularly effective at removing endotoxins (e.g., QXL resin).

Alternatively, cation exchange chromatography may be utilized in amanner such that positively charged solutes bind to the solidchromatographic media and the endotoxin flows through.

Separations using affinity ligands that bind endotoxin or modifiedendotoxin binding ligands are also envisioned. Examples include but arenot limited to histamine, nitrogen-containing heterocyclic compounds, orpolymyxin B.

It is also envisioned to use more than one technique to achievepurification. An example is EndoTrap® column with HPLC/FPLC-automatedsystem. Also useful is endotoxin removal resin that combines porouscellulose beads and an FDA-approved food preservative, poly(ε-lysine),as an affinity ligand to selectively bind endotoxins.

As an alternative to purification or in addition to purification,reduction of non-specific elevation in INFγ levels can be accomplishedby neutralizing endotoxin in the blood to prevent non-specificstimulation. As an example, polymyxin B (PMX) accomplishes this and thenon-specific stimulating effects of contaminating endotoxin are blockedby contacting the blood with PMX before the addition of mKatG or PPD(both containing endotoxin) in their separate conditions. Without PMX,there can be non-specific stimulation of uncertain magnitude which canvary considerably from person to person whether the amount of endotoxincontained in the added reagents is roughly similar or different.

Neutralizing agents for endotoxin are known and all suitable agents areenvisioned, including but not limited to chemicals (e.g.,lipopolyamines), proteins (e.g., human lipopolysaccharide-bindingprotein, hLBP), endotoxin neutralizing peptides (e.g., natural hostdefense peptides, fragments of LPS binding proteins and engineeredpeptides), structural classes of cationic amphiphiles, both peptides andnon-peptidic small molecules. Examples include antimicrobial peptides,such as the skin antimicrobial peptides of the southern bell frog,LPS-binding peptides, such as Li5-001, having the amino acid sequenceKNYSSSISSIHAC, or the dodecapeptide, Li5-025 having amino acid sequenceK′YSSSISSIRAC′, wherein K′ and C′ are D-forms of K and C, respectively(Matsumoto et al., 2010. J. Microbiol. Methods. 82, 54-58). Typicalexamples of endotoxin binding ligands include histamine,nitrogen-containing heterocyclic compounds, and polymyxin B. Alsoincluded are herbs (e.g., Gardenia jasminoides Ellis) or their bioactivecomponents that have endotoxin neutralizing activity (e.g., geniposide).

In general, without added endotoxin neutralizing agent, endotoxinmeasurement in the blood conditions without added reagents should bedown to 0.25-1.0 EU/ml, preferably 0.1-0.25 EU/ml, and more preferablyless than 0.1 EU/ml to be substantially neutralized. Measurement lessthan 0.01 EU/ml is most preferable and is considered endotoxin free.

Endotoxin measurements for PPD preparations should be down to 0.25-1.0EU per microgram of protein, preferably 0.1-0.25 EU per microgram, morepreferably 0.01-0.10 EU per microgram and even more preferably <0.01 EUper microgram to be substantially neutralized. Endotoxin measurements inthe blood condition with PMX added as a neutralizing agent followed byaddition of PPD should be down to 1.5-10 EU per ml, preferably 1.0-1.5EU per ml, more preferably 0.50-1.0 EU/ml, even more preferably0.10-0.50 EU per ml and even more preferably <0.10 EU per ml to besubstantially neutralized. Endotoxin measurements in the blood conditionwith PMX added as a neutralizing agent followed by addition of mKatGshould not be greater than 200 EU per ml, preferably 100-200 EU per ml,more preferably 50-100 EU per ml and even more preferably 10-50 EU perml to be substantially neutralized. Prior purification of mKatG reagentthat results in the level of endotoxin below 10 EU per ml when added tothe whole blood condition may degrade the stimulatory potency of mKatGand are less preferable than 10-50 EU per ml.

There are many sources of endotoxin contamination in the laboratory.Water is perhaps the greatest source of contamination. High purity wateris absolutely essential. Endotoxin can adhere strongly to glassware andplastics unless decontaminated by the inactivation of endotoxin. Otherpotential sources of endotoxin contamination are worker's fingers,chemical reagents, raw materials, and buffers.

If the blood test were performed in a laboratory where unpredictableendotoxin effects were apparent, this would alter the test results anddegrade operating characteristics of the test.

If the blood test is adjusted to optimize receiver operatingcharacteristics (ROC), then the amount (dose) of mKatG and PPD that areadded to each 1-ml condition is subject to change. It is contemplatedthat the sarcoidosis blood test dose for mKatG and PPD results in afinal concentration of 0.1-50 microgram/ml in each respective condition.Preferably, the dose for mKatG and PPD results in a final concentrationof 0.5-20 micrograms/ml. More preferably, the dose for mKatG and PPDresults in a final concentration of 1.0-10 micrograms/ml. Still morepreferably, the dose for mKatG and PPD results in a final concentrationof 1-5 micrograms/ml. Most preferably, the dose for mKatG results in afinal concentration of 2 micrograms/ml, and the dose for PPD results ina final concentration of 5 micrograms/ml.

If the blood test is adjusted to optimize receiver operatingcharacteristics (ROC), then the INFγ levels in the algorithm are subjectto change. In a preferred embodiment, the levels can be adjusted toreach a desired sensitivity and specificity. A positive test forsarcoidosis requires meeting both the specification that mKatG minusbkd>100 pg/ml INFγ and the specification that concentration of INFγ inmKatG condition is greater than the concentration of INFγ in the PPDcondition. Taking the first specification, a slight deviation from thealgorithmic value of 100 would not change the prediction but a largedeviation would. For example, a threshold level of 101 or 99 instead of100 would not materially change the sensitivity or specificity of thetest. Large deviations from the threshold level of 100 (e.g. where mKatGminus bkd>200 for sarcoidosis diagnosis) would materially affect thesensitivity and specificity of the test. In general, levels of INFγ muchgreater than 100 pg/ml would reduce test sensitivity but increase testspecificity for a positive sarcoidosis diagnosis. For example, analgorithm using a level of 200 pg/ml INFγ for mKatG minus background mayincrease specificity to 100% but decrease sensitivity because of thegreater response needed to meet this threshold. In general, levels ofINFγ much lower than 100 pg/ml would reduce test specificity butincrease sensitivity for a positive sarcoidosis diagnosis. It iscontemplated that the algorithm value for mKatG minus background isgreater than 10 pg/ml. More preferably, the algorithm value for mKatGminus background is greater than 80 pg/ml. Still more preferably, thealgorithm value for mKatG minus background is greater than 500 pg/ml.Even more preferably, the algorithm value for mKatG minus background isgreater than 200 pg/ml. Most preferably, the algorithm value for mKatGminus background is greater than 100 pg/ml IFNγ.

The second specification that mKatG>PPD for a sarcoidosis diagnosis isdesigned to differentiate between sarcoidosis and mycobacterial infectedindividuals or those who have had BCG vaccination. A slight deviationfrom this specification on either side of the unequal sign would notchange the prediction. For example, the specification that mKatG isgreater than PPD plus 1 pg/ml INFγ or mKatG plus 1 pg/ml INFγ is greaterthan PPD (i.e., mKatG>PPD minus 1 pg/ml) would not materially affect theresults. A large deviation from this specification would significantlyaffect test characteristics and its diagnostic performance. For example,a specification that a sarcoidosis diagnosis requires mKatG>(PPD+400pg/ml (or more)) will decrease sensitivity but increase specificitysince the higher threshold for mKatG makes it more difficult for asarcoidosis diagnosis. An example that modifies the algorithm tomKatG>(PPD minus 400 pg/ml (or more)) will increase test sensitivity butsignificantly reduce specificity. Deviations between these extremes mayprovide optimal test characteristics. In some examples, mKatG>(PPD+50pg/ml) or mKatG>(PPD minus 20 pg/ml) might produce the optimal testcharacteristics. It is contemplated that the algorithm for a sarcoidosisdiagnosis requires the condition mKatG>PPD+300 pg/ml or the conditionthat mKatG>PPD minus 300 pg/ml. More preferably, it is contemplated thatthe algorithm for a sarcoidosis diagnosis requires the conditionmKatG>(PPD+100 pg/ml) or the condition mKatG>(PPD minus 100 pg/ml). Evenmore preferably, it is contemplated that the algorithm for a sarcoidosisdiagnosis requires the condition mKatG>(PPD+50 pg/ml) or the conditionmKatG>(PPD minus 50 pg/ml). Still more preferably, it is contemplatedthat the algorithm for a sarcoidosis diagnosis requires the conditionmKatG>(PPD+20 pg/ml) or the condition mKatG>(PPD minus 20 pg/ml). Mostpreferably, it is contemplated that the algorithm for a sarcoidosisdiagnosis requires the condition mKatG>PPD.

Thus, taking into consideration variability when conducting testing indifferent laboratories, a more universal algorithm to apply for asarcoidosis diagnosis would use the value that mKatG minus background isgreater than 10 pg/ml IFNγ; and mKatG is greater than PPD+300 pg/ml IFNγor mKatG is greater than PPD minus 300 pg/ml IFNγ.

-   These considerations as to laboratory variability then, as applied    to the entire blood assay test would require a more universal    algorithm that sarcoidosis is indicated when:-   1. mKatG minus background is greater than 10 pg/ml IFNγ.-   2. mKatG is greater than (PPD plus 300 pg/ml IFNγ), or mKatG is    greater than (PPD minus 300 pg/ml IFNγ).-   For this universal algorithm to be have the test characteristics    desired for a diagnostic test, then also:-   3. mKatG amount added to 1 ml blood: ≧0.1 mcg/ml.-   4. PPD amount added to 1 ml blood: ≧0.1 mcg/ml.

The inventive diagnostic blood test for sarcoidosis includes a separatecondition where a T cell stimulating reagent is added to blood toprovide a positive control condition. This condition does not affect theuniversal algorithm described above. The specific type of T cellstimulating reagent used in the assay is not critical as long as thereagent stimulates a large fraction of T cells. Generally, the T cellstimulating reagent should stimulate more than 10% of T cellscirculating in blood of healthy individuals and more than about 20%,30%, 50%, 70% or more of all T cells present. A skilled artisan may usea T cell stimulating reagent including (but not limited to) reagentssuch as toxins that stimulate T cells through binding to specific T cellreceptor proteins (e.g. Staphylococcal enterotoxin B or A), mitogenssuch as phytohaemagglutinin or phorbol myristate acetate (PMA), orantibodies to specific surface receptors on T cells (e.g., anti-CD3) ora combination of reagents. This condition provides an essential qualitycontrol measure and assists in the interpretation of whether anindividual is capable of responding to the other test conditions (mKatG,PPD). For example, a low level of INFγ released in the positive controlwould indicate that a negative test may be the result of incorrect bloodhandling or an individual who is immunosuppressed. In another example,if the level of IFNγ released in the background, mKatG or PPD conditionsapproaches the level of INFγ released in the positive T cell controlcondition, this would lead the test result to be discarded because ofthe possibility of reagent or culture contamination. (Since mKatG andPPD contain a limited number of immune stimulating fragments and thus,would only stimulate a small fraction (<20% and typically much lowerthan 20%) of circulating blood T cells, the release of INFγ in the mKatGand PPD conditions would not be expected to approach the amount of INFγreleased in a positive control condition that stimulates a largefraction of T cells). It is contemplated that if the INFγ released inthe background, mKatG or PPD conditions is greater than 20%, preferably50%, more preferably 60%, most preferably 80% or greater of the positivecontrol, the test result would be discarded with a recommendation torepeat the test.

The mycobacterial catalase-peroxidase protein used in the blood test maybe from various species of mycobacteria (e.g. Mycobacteriumtuberculosis, Mycobacterium smegmatis, Propionibacterium acnes,Helicobacter pylon) or may include active fragments, fusion proteins ormodified protein. Alternative species of the protein are described inU.S. Pat. Pub. No. US 2009/0175798.

In the examples shown below, a full length recombinant mKatG was used inthe blood tests. The recombinant mKatG is slightly modified from theprecise gene sequence of Mycobacterium tuberculosis due to cloning inthe vector which adds amino acids.

Microbial Catalase or Peroxidase Protein Composition

Specific microbial proteins in sarcoidosis tissues, mycobacterialcatalase-peroxidase proteins, are targets of the immune system ofpatients with sarcoidosis. Thus, provided are isolated recombinantand/or purified microbial catalase or peroxidase polypeptides.

In one embodiment, the polypeptide comprises a sequence having at leastabout 90%, preferably about 95%, more preferably about 96%, still morepreferably about 97%, still more preferably about 98%, yet morepreferably about 99% and most preferably about 100% sequence homology tothe sequence of Mycobacterium tuberculosis KatG as described in U.S.Pat. Publication No. US 2009/0175798, or to a fragment thereof, e.g., anantigenic fragment. The mKatG is 740 amino acids in length. A blood testis contemplated that uses large antigenic fragments of mKatG asantigens. The fragments that are contemplated are the fragments of aminoacids 1-631, preferably amino acids 1-672 and most preferably aminoacids 1-705. In another embodiment the contemplated fragment is aminoacids 5-470 and preferably amino acids 5-631. These ranges include alarge majority of potential antigenic peptides within the full lengthmKatG. For example, it is likely that a portion of the full length mKatGthat contains 90% of peptide fragments known to bind to some polymorphicMHC molecules would provide sufficient antigenic stimulation in 90% orso of sarcoidosis patients.

Blood Test as Biomarker

The inventive blood test described in this application may also serve asa prognostic tool to predict the likelihood of the subsequent clinicalcourse of sarcoidosis, for example, whether the course of sarcoidosishas undergone remission (where the inflammation subsides andanti-inflammatory treatment is not needed) or whether the sarcoidosis ischronic with persistent or progressive disease. Further included isusing the inventive blood test as a monitor of disease “activity”.Active disease is generally meant to include persistent or worseningsymptoms and/or laboratory or clinical imaging studies that indicate thepresence of ongoing or progressive inflammation.

In one embodiment, individuals with sarcoidosis who have a positivediagnostic blood test on initial testing for sarcoidosis will have arepeat test in follow-up during their clinical course. Those individualswho are not on treatment and have a negative test (mKatG minus bkd<100)would predict that the disease is in remission and does not needtreatment. In this situation, if this blood test turns positive (mKatGminus bkd>100) in further future testing, this would indicate a returnof active disease. In another embodiment, this inventive blood test canbe used to assess whether a prescribed treatment (using therapiesincluding but not limited to corticosteroids, immunosuppressive andanti-TNF therapies) is effective and being used in an adequate dose tosuppress disease activity. For those individuals who are on treatment(and had a positive initial blood test), a negative blood test (mKatGminus bkd<100) indicates that treatment is currently adequate insuppressing disease activity. Individuals with sarcoidosis who are beingtreated and have a positive repeat blood test (mKatG minus bkd>100)indicates the treatment is ineffective or being used in an inadequatedose. This inventive blood test may be particularly useful insarcoidosis to assess adequacy of treatment when patients are beingtapered on their corticosteroid or other anti-inflammatory treatmentsi.e., a positive test indicates active disease that needs additionaltreatment, whereas a negative test supports the adequacy of the currentlevel of treatment.

If the inventive blood test used for these purposes is adjusted tooptimize receiver operating characteristics (ROC), then the INFγ levelsin the algorithm are subject to change. For the use of this blood testfor these purposes, it is contemplated that the algorithm value formKatG minus background is greater than 10 pg/ml IFNγ. More preferably,the algorithm value for mKatG minus background is greater than 80 pg/mlIFNγ. Still more preferably, the algorithm value for mKatG minusbackground is greater than 500 pg/ml IFNγ. Even more preferably, thealgorithm value for mKatG minus background is greater than 200 pg/mlIFNγ. Most preferably, the algorithm value for mKatG minus background isgreater than 100 pg/ml IFNγ.

Such a test may be employed at multiple times during the clinical courseof sarcoidosis. This test may be used together with other patientinformation derived from tests including but not limited to genetictests, proteomic profiles of tissues or blood, or other tests of generalimmunity in sarcoidosis patients, in order to enhance the testcharacteristics as a diagnostic tool or as an aid in clinicalmanagement.

The following examples set forth the general procedures involved inpracticing the present invention. To the extent that specific materialsare mentioned, it is merely for purposes of illustration and is notintended to limit the invention.

EXAMPLE 1

Blood Test Methodology

Patients with biopsy-proven sarcoidosis and control subjects (includingPPD+, BCG+ controls and subjects undergoing bronchoscopy) were recruitedwith informed consent and IRB approval. A whole blood stimulationINFγ-release assay was tested using full-length recombinant (rec)-mKatGand PPD as antigens. INFγ-release after 24 hrs was measured by ELISA ineach condition and in a separate background control condition in whichculture media was added. Staphylococcal enterotoxin B (Toxin Technology)was used as a positive control. Following pilot studies assessingoptimal doses and conditions, a sarcoidosis diagnosis was determined bythe following results: mKatG minus media (bkd)>100 pg/ml and mKatG>PPD.

Material and Methods

Study Population

Clinical samples were obtained from patients with sarcoidosis, healthysubjects, patients with non-sarcoidosis lung disease or other systemicinflammatory diseases recruited from specialized clinics or hospitals ofthe Johns Hopkins University. A diagnosis of sarcoidosis was establishedeither by tissue biopsy or by initial manifestations consistent withLofgren syndrome (erythema nodosum and/or acute arthritis, hilarlymphadenopathy) without alternative diagnoses according to world-wideconsensus criteria. Based on clinical manifestations, chest radiograph,and pulmonary function tests, patients were classified as having activesarcoidosis or “inactive” disease, defined by resolution of diseasemanifestations or absence of disease progression off all therapy for atleast 1 year. Untreated patients were those who had not receivedsystemic therapy within 3 months of the time of study. Control subjectsincluded healthy individuals with documented skin testing to purifiedprotein derivative (PPD) within the past year or with a self-reportedprior history of BCG vaccination. PPD skin testing was performed inaccordance with accepted criteria used in the respective countries. Allstudy subjects participated voluntarily and provided informed consentunder protocols approved by the local institutional review board.

Reagents

Complete medium was made from RPMI (Cellgro Mediatech Inc.), 10% pooledhuman AB serum (Sigma-Aldrich), 1% penicillin-streptomycin (Biosource),1% Sodium Pyruvate (Sigma), 1% Non-essential amino acids (Gibco), 2.5%Hepes buffer (Quality Biological).

Recombinant Mtb KatG protein was isolated and prepared using an E. coliUM255 strain overexpression system carrying a plasmid construct pYZ56containing the wild-type M. tuberculosis katG gene in a 2.9 kDEcoRV-KpnI fragment in pUC19 vector (Zhang et al, Nature (1992)358:591-593) and as published in Chen et al. J Immunol. (2008);181:8784-96. PMID: 19050300. The culture was grown in LB mediumcontaining 100 μg/ml ampicillin and agitated overnight at 37° C. Thecells were harvested by centrifugation at 4000 g for 15 min at 4° C.Cell pellets were resuspended in 100 ml of 10 mM phosphate buffer(Na₂HPO4 and NaH₂PO₄ and 0.5 mM EDTA) (pH 6.0) and sonicated with three30 s bursts at full power. Insoluble material was removed bycentrifugation at 12000 g, 4° C. for 30 min. The supernatant washarvested for further purification by ammonium sulfate precipitation,and the protein was harvested by centrifugation at 12000 g for 30 min.The pellet was resuspended in the phosphate buffer and dialyzed againstthe same buffer at 4° C. overnight and then assayed for peroxidase andcatalase activity. The active fractions were further purified by gelfiltration chromatography. A SUPERDEX® 200 gel filtration column(Pharmacia) was equilibrated with the phosphate buffer overnight. Thecatalase containing fractions were loaded onto the column with a flowrate of 0.2 ml/min. Fractions (1 ml) were collected, and assayed forperoxidase and catalase activity. Active fractions were assessed forpurity by SDS-PAGE, pooled, and then dialyzed against the abovephosphate buffer at 4° C. overnight (Johnsson, K. et al. J Biol Chem(1997) 272:2834-2840). The purified KatG protein was at least 95% pure.The protein was kept at −80° C. for long term storage and −20° C. forshort term (<2 months) between immunological studies.

PPD was obtained from Staten Serum Institut. The PPD was furtherpurified by EndoTrap® Endotoxin Removal Kit (Hyglos, Germany) using 3flow through passes following manufacturer's recommendations.

Staphylococcal enterotoxin B (SEB) was purchased from Toxin Technology.Cells were stimulated with either recombinant mKatG or PPD (Staten SerumInstitut), or with Staphylococcal enterotoxin B (SEB) (Toxin Technology)as a positive control.

Whole Blood IFNγ Release Assay

Briefly, whole blood was obtained by phlebotomy and placed into aheparinized tube. The blood was mixed by pipetting up and down 5 times,and then 1 ml aliquots of whole blood were added to individual 5 mlpolypropylene snap-cap round bottom tubes. Reagents were added toindividual tubes: 10 μl of complete media (or no added media), PMX final10 μg/ml, mKatG plus PMX 10 μg/ml, PPD plus PMX 10 μg/ml and SEB 1μg/ml. The tubes were lightly vortexed and incubated at 37° C. in ahumidified CO₂ incubator for 24 hr. with loose snap caps. After 24 hrs,the plasma layer was harvested by pipette, transferred to microfugetubes with 25-40 μl of EDTA per plasma sample, centrifuged 1000×g for 3minutes to pellet blood cells, the plasma transferred to a second set ofmicrofuge tubes with 20 μl of EDTA, centrifuged again and then theplasma was transferred to a clean microfuge for storage at −80 degCelsius until measurement of INFγ levels. INFγ levels were measured byELISA (BioLegend) following manufacturer's protocol.

Statistics

Statistical analyses were performed Fisher's exact test or withchi-square analysis and ROC curve generation was performed usingGraphPad Prism 5 (GraphPad Software).

Results

TABLE 1 rec-mKatG/PPD

*mKatG near SEA/SEB positive controls, presumed contamination **insertedby mistake in original presentation

TABLE 2 SUMMARY OF ASSAY RESULTS Test Results rec mKatG/PPD assay PosNeg Total Sensitivity Specificity Active Sarcoidosis, 20 11 31 65%untreated Controls 1 40 41 98% BCG+, PPD+ or NTM 0 16 16 100%

We explored the operating characteristics of this test usingrecombinant-mKatG and PPD. Using 2 μg/ml recombinant-mKatG and criteriaabove, 20/31 (65%) sarcoidosis patients were positive for a sarcoidosisdiagnosis vs. 1/41 (98%) controls (Fisher's exact test, p<0.0001). All16 BCG+ or PPD+ subjects or patients with non-tuberculous mycobacterialinfection were negative for a sarcoidosis diagnosis. These data indicatethe test has a sensitivity of 65%, a specificity of 98%, a positivepredictive value of 95%, a negative predictive value of 79% and alikelihood ratio of 26.45. The confidence interval for the positivepredictive value of this test is 0.7618 to 0.9988.

These results suggest a whole blood serum INFγ-release assay using mKatGand PPD has a high positive predictive value for sarcoidosis.

EXAMPLE 2

Processing of Whole Blood Samples for 24 hr Plasma Collection

-   1. Label all sterile polypropylene cell culture tubes with Subject    No. and condition.-   2. Uncap 1 heparinized tube of whole blood. Pipet up and down (5×)    with an individually wrapped sterile 5 ml serological pipet for    mixing.-   3. Set up sterile 5 ml polypropylene, snap-cap, round bottom tubes.-   4. Add 1 ml whole blood from heparinized tube using individually    wrapped 1 ml sterile serological pipet directly to each respective    empty tube.-   5. Add reagents as specified below to appropriate tubes beginning    with PMX first, followed by mKatG, PPD and then Staphylococcal    enterotoxin B (SEB).

Test Conditions:

-   A. No addition (bkd)-   B. PMX 10 μg/ml-   C. mKatG (optimal dose(s) may vary dependent on test conditions; in    experiments shown here: (2 μg/ml)+PMX 10 μg/ml (added first).-   D. PPD (5 μg/ml)+PMX 10 μg/ml (added first).-   E. SEB-(1 μg/ml) 1 μl from stock (positive control)-   6. Lightly (pulse) vortex each tube to mix whole blood. Place all    test conditions in 37° C./5% CO₂ incubator for 24 hrs with loose    snap-caps.-   7. After 24 hrs, remove tubes from incubator and note the plasma    layer residing above the cellular layer of blood. Leave plasma layer    undisturbed.-   8. PLASMA HARVEST and TRANSFER: 2 transfers to clean the plasma    before storage:-   9. Set up two sets of microcentrifuge tubes in a tube rack, numbered    1-10. The two sets are for sequential transfers of the plasma.-   10. Add 25-40 μl (1:10 EDTA per plasma sample) of 20 mM EDTA to the    2^(nd) set of microcentrifuge tubes for the final transfer.-   11. Carefully transfer plasma (using a 200 μl pipet) usually 2 pipet    fills of 200 82 l or more) from the original stimulation tube to the    1^(st) set of 1.5 ml microcentrifuge tubes. (Avoid drawing blood    into the plasma). Collect the “clean” plasma, an average of 300-500    μl.-   12. Spin microcentrifuge tubes at 1000×g for 3 minutes.-   13. Transfer plasma, (leave whole blood pellet undisturbed) into the    2^(nd) set of microcentrifuge tubes with the EDTA. (the final    concentration of EDTA is about 2 mM and prevents clotting in the    samples).-   14. Store samples at −80° C.-   15. For subsequent ELISA runs, dilute the thawed samples 1:4 with    the ELISA diluent for INFγ. Discard any clots that may form in the    samples.

Each sample is measured for concentration of INFγ by ELISA (INFγ ELISAkit, BioLegend, San Diego, Calif.).

Reagents for use in the above procedure:

-   1. none-   2. PMX (Polymyxin B; Sigma-Aldrich) commercially available.-   3. recombinant mKatG prepared as described in: Chen E S, et al. J    Immunol. 2008; 181:8784-96.-   4. Purified protein derivative (PPD) (from Staten Serum Institut,    Denmark). This is further purified using commercially purchased    Endotrap® columns to reduce endotoxin levels to <0.10 EU/microgram.-   5. Staphylococcal enterotoxin B (SEB) commercially purchased,    positive control.

The algorithm used to compare the results is the following: the INFγconcentration in the mKatG condition minus the INFγ concentration in thebackground condition is greater than 100 pg/ml and the INFγconcentration in the mKatG condition is greater than the INFγconcentration in the PPD condition. For a positive test for sarcoidosis,both specifications must be present. Otherwise, the result isnondiagnostic.

In the experiments described above, an mKatG dose of 2 μg/ml and a PPDdose of 5 μg/ml, and the cut-off thresholds provided in the algorithm(INFγ levels of mKatG minus background>100 pg/ml and mKatG>PPD for apositive test for sarcoidosis) optimize the positive predictive value ofthe blood test

REFERENCES

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

What is claimed is:
 1. A method for testing for sarcoidosis comprisingcontacting a first aliquot of whole blood with medium alone, a secondaliquot of whole blood with mKatG, a third aliquot of whole blood withPPD, a fourth aliquot of whole blood with a T cell stimulating reagent;incubating the aliquots about 12 to 24 hours; determining theconcentration of INFγ in the whole blood combinations tested; andevaluating the INFγ concentrations using the algorithm that the blood ispositive for sarcoidosis if the concentration of INFγ from whole bloodcontacted with mKatG minus whole blood contacted with no added reagentis greater than 100 pg/ml and the concentration of INFγ from whole bloodcontacted with mKatG is greater than the concentration of INFγ for wholeblood contacted with PPD.
 2. A method for testing for sarcoidosiscomprising: a. combining aliquots of whole blood separately withdifferent reagents or with a control of no reagent; wherein thecombinations formed are whole blood plus: i. background (no addedstimulatory reagent), ii. PMX, iii. mKatG plus PMX, iv. PPD plus PMX,and v. T cell stimulating agent; b. testing a sample from each of thealiquot combinations of step a for concentration of INFγ; and; c.applying the algorithm that the blood is positive for sarcoidosisdiagnosis if the concentration of INFγ for mKatG minus background isgreater than 100 pg/ml and the concentration of INFγ for mKatG isgreater than the concentration of INFγ for PPD.
 3. A method fordetermining whether an individual has or is likely to developsarcoidosis, comprising contacting and incubating first immune cellsfrom whole blood of the individual with a microbial catalase orperoxidase protein; contacting and incubating second immune cells fromwhole blood of the individual with PPD, wherein contaminating endotoxinsare substantially absent or substantially neutralized in the contactingand incubating steps; and measuring INFγ release following theincubating step; wherein when the concentration of INFγ inmKatG-stimulated blood minus background is greater than 100 pg/ml andthe concentration of INFγ in mKatG-stimulated blood is greater than theconcentration of INFγ in PPD-stimulated blood, the individual has or islikely to develop sarcoidosis.
 4. A kit for diagnosing sarcoidosiscomprising a first composition comprising a microbial catalase orperoxidase protein, a second composition comprising purified proteinderivative and optionally instructions for use.
 5. A method forpredicting sarcoidosis by results of a blood test comprising: a.treating whole blood aliquots according to different conditionscomprising: i. no addition, ii. addition of mKatG, iii. addition of PPD,iv. addition of a T cell stimulating reagent; b. measuring for INFγreleased by the treated blood cells; and c. predicting sarcoidosis ifthe measurement of INFγ in mKatG-treated blood is greater than theconcentration of INFγ in PPD-treated blood and the measurement of INFγin mKatG-treated blood minus background is greater than 100 pg/ml.
 6. Amethod for aiding in the prediction of whether an individual hassarcoidosis, the method comprising: (a) treating a first aliquot ofblood from the individual as a control having no added INFγ-releasingreagent; (b) contacting a second aliquot of blood from the individualwith fluid containing mKatG in an amount that is ≧0.1 mcg/ml; (c)contacting a third aliquot of blood from the individual with fluidcontaining PPD in an amount that is ≧0.1 mcg/ml; (d) detecting theamount of INFγ in the aliquots; (e) calculating adjusted amounts of INFγas amounts of INFγ in the second and third aliquots minus the amount ofINFγ in the first aliquot; and (f) associating a prediction ofsarcoidosis with the determination that there is (1) an adjusted amountof INFγ for the second aliquot of greater than 100 pg/ml as well as thatthere is (2) an adjusted amount of INFγ for the second aliquot that isgreater than the adjusted amount of INFγ for the third aliquot.
 7. Themethod of claim 6 further comprising the steps of contacting a fourthaliquot of blood from the individual with a control fluid containing aT-cell stimulating reagent, detecting the level of INFγ in the fourthaliquot, calculating an adjusted level of INFγ as the level of INFγ inthe fourth aliquot minus the level of INFγ in the first, second andthird aliquot; and associating a prediction of a quality controlspecification.
 8. The method of claim 6 wherein the detecting isperformed by the ELISA procedure.
 9. The method of claim 6 wherein thePPD reagent used in the blood test is substantially free of endotoxin toa level of at most 0.25-1.0 EU/microgram protein and when used in theblood test results in a level of endotoxin<10 EU/ml.
 10. The method ofclaim 6 where the mKatG reagent has been purified so that when used inthe blood test results in a level of endotoxin of 10-200 EU/ml.
 11. Amethod to distinguish between individuals with sarcoidosis andindividuals with tuberculosis, individuals previously vaccinated withBCG, individuals with diseases other than sarcoidosis, or healthyindividuals comprising the steps of: a. exposing aliquots of a bloodsample with a non-stimulatory control, an isolated microbial catalase orperoxidase protein or antigenic fragments thereof, isolated PPD orantigenic fragments thereof, and a T-cell stimulatory control; b.assaying said exposed aliquots for the release of INFγ; c. using saidsample exposed to a T-cell stimulatory control as a positive control forIFNγ detection and as a control for reagent contamination whereas thesample is discarded if said concentration of INFγ detected in saidsample exposed to a microbial catalase or peroxidase protein orantigenic fragments thereof is greater than 50% of the concentration ofINFγ detected in said sample exposed to a T-cell stimulatory control; d.normalizing the concentration of INFγ in said sample exposed to amicrobial catalase or peroxidase protein or antigenic fragments thereofby subtracting the concentration of INFγ detected in said sample exposedto said non-stimulatory control; and e. determining whether saidindividual is positive for sarcoidosis whereas both conditions aresatisfied for the algorithm comprising:
 1. the normalized concentrationof INFγ detected in said sample exposed to a microbial catalase orperoxidase protein or antigenic fragments thereof is greater than theconcentration of a laboratory specific threshold wherein said thresholdis the concentration of INFγ that is greater than the concentrationnormalized as in step d., detected in 70% of control subjects that arenegative for sarcoidosis and negative for mycobacterial disease, and 2.wherein the concentration of INFγ detected in said sample exposed to amicrobial catalase or peroxidase protein or antigenic fragments thereofminus the concentration of INFγ detected in said sample exposed to PPDor antigenic fragments thereof is greater than the same value detectedin greater than 90% of mycobacterial infected individuals.
 12. A methodto distinguish between individuals with sarcoidosis and individuals withtuberculosis, individuals previously vaccinated with BCG, individualswith diseases other than sarcoidosis, or healthy individuals comprisingthe steps of: a. exposing aliquots of a blood sample with anon-stimulatory control, an isolated microbial catalase or peroxidaseprotein or antigenic fragments thereof, isolated PPD or antigenicfragments thereof, and a T-cell stimulatory control; b. assaying saidexposed aliquots for the release of INFγ; c. using said sample exposedto a T-cell stimulatory control as a positive control for IFNγ detectionand as a control for reagent contamination whereas the sample isdiscarded if said concentration of INFγ detected in said sample exposedto a microbial catalase or peroxidase protein or antigenic fragmentsthereof is greater than 50% of the concentration of INFγ detected insaid sample exposed to a T-cell stimulatory control; d. normalizing theconcentration of INFγ in said sample exposed to a microbial catalase orperoxidase protein or antigenic fragments thereof by subtracting theconcentration of INFγ detected in said sample exposed to saidnon-stimulatory control; and e. determining whether said individual ispositive for sarcoidosis whereas both conditions are satisfied for thealgorithm comprising:
 1. the normalized concentration of INFγ detectedin said sample exposed to a microbial catalase or peroxidase protein orantigenic fragments thereof is greater than the concentration of alaboratory specific threshold wherein said threshold is theconcentration of INFγ that is greater than the concentration normalizedas in step d., detected in 70% of control subjects that are negative forsarcoidosis and negative for mycobacterial disease, and
 2. wherein theconcentration of INFγ detected in said sample exposed to a microbialcatalase or peroxidase protein or antigenic fragments thereof is greaterthan the concentration of INFγ detected in said sample exposed to PPD orantigenic fragments thereof plus or minus 300 pg/ml.
 13. A method todistinguish between individuals with sarcoidosis and individuals withtuberculosis, individuals previously vaccinated with BCG, individualswith diseases other than sarcoidosis, or healthy individuals comprisingthe steps of: a. exposing aliquots of a blood sample with anon-stimulatory control, an isolated microbial catalase or peroxidaseprotein or antigenic fragments thereof, isolated PPD or antigenicfragments thereof, and a T-cell stimulatory control; b. assaying saidexposed aliquots for the release of INFγ; c. using said sample exposedto a T-cell stimulatory control as a positive control for IFNγ detectionand as a control for reagent contamination; d. normalizing theconcentration of INFγ in said sample exposed to a microbial catalase orperoxidase protein or antigenic fragments thereof by subtracting theconcentration of INFγ detected in said sample exposed to saidnon-stimulatory control; and e. determining whether said individual ispositive for sarcoidosis whereas both conditions are satisfied for thealgorithm comprising:
 1. the normalized concentration of INFγ detectedin said sample exposed to a microbial catalase or peroxidase protein orantigenic fragments thereof is greater than the concentration of adiagnostic cut-off level determined by testing control subjects that arenegative for sarcoidosis and negative for mycobacterial disease and thatcan be adjusted to meet desired levels of specificity and sensitivity,and
 2. wherein the concentration of INFγ detected in said sample exposedto a microbial catalase or peroxidase protein or antigenic fragmentsthereof is greater than the concentration of INFγ detected in saidsample exposed to PPD or antigenic fragments thereof plus or minus Y,whereas Y is a variable determined by testing a combination of controlsubjects, including individuals with mycobacterial infections,non-sarcoidosis individuals and healthy individuals, and that can beadjusted to meet desired levels of specificity and sensitivity.
 14. Amethod to distinguish between individuals with sarcoidosis andindividuals with tuberculosis, individuals previously vaccinated withBCG, individuals with diseases other than sarcoidosis, or healthyindividuals comprising the steps of: a. exposing aliquots of a bloodsample with a non-stimulatory control, an isolated microbial catalase orperoxidase protein or antigenic fragments thereof, isolated PPD orantigenic fragments thereof, and a T-cell stimulatory control; and b.preparing said aliquots for further analysis.
 15. The method of claim 14further comprising assaying said exposed aliquots for the release ofINFγ.
 16. The method of claim 15 further comprising: a. using saidsample exposed to a T-cell stimulatory control as a positive control forIFNγ detection and as a control for reagent contamination; b.normalizing the concentration of INFγ in said sample exposed to amicrobial catalase or peroxidase protein or antigenic fragments thereofby subtracting the concentration of INFγ detected in said sample exposedto said non-stimulatory control; and c. determining whether saidindividual is positive for sarcoidosis whereas both conditions aresatisfied for the algorithm comprising:
 1. the normalized concentrationof INFγ detected in said sample exposed to a microbial catalase orperoxidase protein or antigenic fragments thereof is greater than theconcentration of a diagnostic cut-off level determined by testingcontrol subjects that are negative for sarcoidosis and negative formycobacterial disease, and that can be adjusted to meet desired levelsof specificity and sensitivity, and
 2. wherein the concentration of INFγdetected in said sample exposed to a microbial catalase or peroxidaseprotein or antigenic fragments thereof is greater than the concentrationof INFγ detected in said sample exposed to PPD or antigenic fragmentsthereof plus or minus Y, whereas Y is a variable determined by testing acombination of control subjects, including individuals withmycobacterial infections, non-sarcoidosis individuals and healthyindividuals, and that can be adjusted to meet desired levels ofspecificity and sensitivity.
 17. A method of adapting a blood sample fordiagnosing individuals with sarcoidosis comprising the steps of: a.administering to aliquots of a blood sample a non-stimulatory control,an isolated microbial catalase or peroxidase protein or antigenicfragments thereof, isolated PPD or antigenic fragments thereof, and aT-cell stimulatory control; b. detecting the release of INFγ from saidaliquots wherein said aliquot exposed to a T-cell stimulatory control isused as a positive control for IFNγ detection and as a control forreagent contamination; c. normalizing the concentration of INFγ in saidaliquot exposed to a microbial catalase or peroxidase protein orantigenic fragments thereof by subtracting the concentration of INFγdetected in said aliquot exposed to said non-stimulatory control; and d.determining a diagnosis of sarcoidosis whereas both conditions aresatisfied for the algorithm comprising:
 1. the normalized concentrationof INFγ detected in said aliquot exposed to a microbial catalase orperoxidase protein or antigenic fragments thereof is greater than theconcentration of a diagnostic cut-off level determined by testingcontrol subjects that are negative for sarcoidosis and negative formycobacterial disease, and that can be adjusted to meet desired levelsof specificity and sensitivity, and
 2. wherein the concentration of INFγdetected in said aliquot exposed to a microbial catalase or peroxidaseprotein or antigenic fragments thereof is greater than the concentrationof INFγ detected in said sample exposed to PPD or antigenic fragmentsthereof plus or minus Y, whereas Y is a variable determined by testing acombination of control subjects, including individuals withmycobacterial infections, non-sarcoidosis individuals and healthyindividuals, and that can be adjusted to meet desired levels ofspecificity and sensitivity.
 18. A method of adapting a blood sample fortreating individuals who may have sarcoidosis comprising the steps of:a. administering to aliquots of a blood sample a non-stimulatorycontrol, an isolated microbial catalase or peroxidase protein orantigenic fragments thereof, isolated PPD or antigenic fragmentsthereof, and a T-cell stimulatory control; b. detecting the release ofINFγ from said aliquots wherein said aliquot exposed to a T-cellstimulatory control is used as a positive control for IFNγ detection andas a control for reagent contamination; c. normalizing the concentrationof INFγ in said aliquot exposed to a microbial catalase or peroxidaseprotein or antigenic fragments thereof by subtracting the concentrationof INFγ detected in said aliquot exposed to said non-stimulatorycontrol; and d. treating an individual with a sarcoidosis treatmentstrategy whereas both conditions are satisfied for the algorithmcomprising:
 1. the normalized concentration of INFγ detected in saidaliquot exposed to a microbial catalase or peroxidase protein orantigenic fragments thereof is greater than the concentration of adiagnostic cut-off level determined by testing control subjects that arenegative for sarcoidosis and negative for mycobacterial disease, andthat can be adjusted to meet desired levels of specificity andsensitivity, and
 2. wherein the concentration of INFγ detected in saidaliquot exposed to a microbial catalase or peroxidase protein orantigenic fragments thereof is greater than the concentration of INFγdetected in said sample exposed to PPD or antigenic fragments thereofplus or minus Y, whereas Y is a variable determined by testing acombination of control subjects, including individuals withmycobacterial infections, non-sarcoidosis individuals and healthyindividuals, and that can be adjusted to meet desired levels ofspecificity and sensitivity.
 19. A method for treating individuals whomay have sarcoidosis comprising: treating a patient with a sarcoidosistreatment strategy when receiving a positive result from a diagnostictest wherein said diagnostic test included the step of exposing aliquotsof a blood sample with a non-stimulatory control, an isolated microbialcatalase or peroxidase protein or antigenic fragments thereof, isolatedPPD or antigenic fragments thereof, and a T-cell stimulatory control.20. A method for treating individuals who may have sarcoidosiscomprising treating a patient with a sarcoidosis treatment strategy whenreceiving a positive result from a diagnostic test wherein saiddiagnostic test included the steps of: a. administering to aliquots of ablood sample a non-stimulatory control, an isolated microbial catalaseor peroxidase protein or antigenic fragments thereof, isolated PPD orantigenic fragments thereof, and a T-cell stimulatory control; b.detecting the release of INFγ from said aliquots wherein said aliquotexposed to a T-cell stimulatory control is used as a positive controlfor IFNγ detection and as a control for reagent contamination; c.normalizing the concentration of INFγ in said aliquot exposed to amicrobial catalase or peroxidase protein or antigenic fragments thereofby subtracting the concentration of INFγ detected in said aliquotexposed to said non-stimulatory control; and d. determining a positiveresult whereas both conditions are satisfied for the algorithmcomprising:
 1. the normalized concentration of INFγ detected in saidaliquot exposed to a microbial catalase or peroxidase protein orantigenic fragments thereof is greater than the concentration of adiagnostic cut-off level determined by testing control subjects that arenegative for sarcoidosis and negative for mycobacterial disease, andthat can be adjusted to meet desired levels of specificity andsensitivity, and
 2. wherein the concentration of INFγ detected in saidaliquot exposed to a microbial catalase or peroxidase protein orantigenic fragments thereof is greater than the concentration of INFγdetected in said sample exposed to PPD or antigenic fragments thereofplus or minus Y, whereas Y is a variable determined by testing acombination of control subjects, including individuals withmycobacterial infections, non-sarcoidosis individuals and healthyindividuals, and that can be adjusted to meet desired levels ofspecificity and sensitivity.
 21. A kit for diagnosing sarcoidosiscomprising: a. a first composition comprising a microbial catalase orperoxidase protein or antigenic fragments thereof; b. a secondcomposition comprising purified protein derivative; c. a thirdcomposition comprising a T-cell stimulatory control; and d. optionallyinstructions for use.
 22. A system for diagnosing sarcoidosiscomprising: a. a first composition comprising a purified microbialcatalase or peroxidase protein or antigenic fragments thereof that isconfigured to induce a specific T-cell immune response; b. a secondcomposition comprising purified protein derivative that is configured toinduce a specific T-cell immune response; c. a third compositionconfigured to allow a whole blood sample to be incubated with the firstor second composition; and d. a fourth composition configured to detectthe immune response elicited upon incubation of the first or secondcomposition with whole blood.